It is estimated that there are 8 to 10 million new cases of pulmonary tuberculosis causing approximately 3 million deaths per year, worldwide, making tuberculosis one of the foremost causes of death due to infection. Mycobacterium tuberculosis, the etiological agent of tuberculosis, is an acid-fast, non-motile, rod shaped bacterium. As a result of recent increases in the number of immunocompromised and immunosuppressed patients, MOTT infections are also increasing. For example, infections by M. avium complex (MAC), M. fortuitum, M. chelonae, M. kansasii and several other nontuberculosis mycobacteria referred to as atypical mycobacteria or MOTT are opportunistic pathogens in patients infected with HIV as well as in other immune compromised patients. MOTT species are the etiological agents of chronic pulmonary disease, lymphadenitis, skin and soft-tissue infections, and opportunistic infections in man. MOTT are present in the environment and infect animals as well as humans. Unlike the M. tuberculosis (MTB) complex (M. tuberculosis, M. africanum, M. microtii and M. bovis), molecular methods for rapid detection and identification of MOTT species do not exist.
Conventional methods for the diagnosis of mycobacterial infections involve direct acid-fast staining and organism cultivation, followed by biochemical and morphological assays to confirm the presence of mycobacteria and identify the species. Typical diagnostic methods using conventional culture methods are time-consuming and can take as long as six weeks. Automated culturing systems such as the BACTEC™ system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) can decrease the detection time of mycobacteria to one or two weeks. However, once detected, culturing these slow-growing microorganisms in the presence of antibiotics to determine their drug susceptibility requires several additional weeks. Therefore, a need to further reduce the time required to diagnose mycobacterial infections to provide prompt treatment of mycobacterial infections exists.
Because of important clinical significances of MOTT, it is desirable to develop a method that can quickly and efficiently diagnose and differentiate MTB and MOTT. Unfortunately, the ability to quickly diagnose MOTT in the early stages of infection based on clinical testing is lacking. Presently, a combination of clinical findings and identification of acid-fast bacteria by microscopy in patient samples are by far the most rapid and cost-effective detection methods. However, these tests yield poor sensitivity and specificity and definitive diagnosis by culture is still particularly difficult to determine quickly because it takes about 2 to 8 weeks to grow the culture and gather all data (Springer et al., 1996 J. Clin. Microbiol. 34:296–303 and Wayne et al., 1991). Moreover, some mycobacterial isolates cannot be accurately identified using standard biochemical test alone. Gas chromatography and high performance liquid chromatography (HPLC) provide an accurate identification but often require culture isolates or larger numbers of bacilli (Ramos, 1994 J. Chromatgr. 32:219–227). A commercially available non-isotopic Accuprobe method (GEN-PROBE, Inc.) provides species-specific oligonulceotides probes that hybridize against the RNA of M. avium, M. intracellulare, M. gordonae and M. kansaii. However, this test is only applicable on cultures and cannot be used directly on patient samples (Lebrun et al., 1994 J. Clin. Microbiol. 30:2476–2478).
Molecular methods that provide quick and rapid diagnosis of MOTT in clinical specimens are not available. Available PCR-based methods for diagnosing mycobacterial infections often require considerable time or dedicated equipment for a single test (Yule, 1994 Biotechnol. 12:1335–7 and Eisenach et al., 1991 Am. Rev. Respir. Dis. 144:1160–3). Methods for identifying rapidly growing mycobacteria using restriction fragment length polymorphism (RFLP) of MOTT DNA and other techniques involving complex methodology are not suitable for clinical testing environments (Telenti et al., 1993 J. Clin. Microbiol. 31:175–8; Roth et al, 2000 J. Clin. Microbiol 38:1094–1104; Ringuet et al., 1999 J. Clin. Microbiol 37:852–857 and Avaniss-Aghajani et al., 1996 J. Clin. Microbiol 34:98–102). Further, methods such as multiplex PCR-based assays followed by reverse cross-blot hybridization (Kox et at., 1997 J. Clin. Microbiol. 35:1492–1498) or methods that differentiate mycobacteria species by amplifying the superoxide dismutase gene (Zolg et al., 1997 J. Clin. Microbiol. 32:2801–2812) used for identifying mycobacteria have several clinical disadvantages. In particular, methods requiring hybridization of nucleic acids extracted from patient samples against species-specific probes can only recognize a specific species of MOTT and require large sample volumes or quantities. Moreover, species-specific methods designed for detecting MTB and M. avium in clinical samples (Stauffer et al., 1998 J. Clin. Microbiol. 36:614–617 and Emler et al., 2001 J. Clin. Microbiol. 39:2687–2689) or PCR amplification techniques for differentiating M. avium and M. intracellulare (Chen et al., 1996 J. Clin. Microbiol. 34:1267–1269 and Kulski et al, 1995 J. Clin. Microbiol. 33:668–674), have limited diagnostic utility because these methods differentiate only two species. Therefore, a molecular method capable of diagnosing several MOTT species in both fresh and archival tissue samples in a single test is needed. The present invention not only provides a more rapid method for detecting and differentiating MOTT and MTB, it also significantly decreases the waiting time for growing culture isolates and eliminates the requirement for larger numbers of bacilli.
Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.